The present invention relates to liquid chromatography packing materials, and more particularly it relates to an improved packing material for use in high performance liquid chromatography and to a method for making and using such a packing material.
The application of modern liquid chromatographic techniques by high performance liquid chromatography (HPLC) has led to an improvement of separation, characterization, and purification of proteins. The separations are based on native properties of the proteins like size (or molecular weight), charge at a given pH and hydrophobicity (originating from amino acid composition and tertiary structure). These differences have been used in classical protein separation procedures as well as with chromatographic techniques. The differences in charge are used in electrophoresis and in ion exchange chromatography. Size differences are utilized in ultra centrifuge separation and in size exclusion chromatography (SEC). The differences in hydrophobicity of the individual proteins have been widely applied in fractionated precipitation by the addition of neutral salts. The chromatographic separation with hydrophobic stationary (reversed) phases has been proven to be a highly effective and versatile tool for protein separation and characterization.
Liquid chromatography using a reverse phase packing has been found to be an effective tool in both qualitative and quantitative analysis for drug substances in blood, serum, or plasma. Typically the reverse phase packing material is made up of bonded alkyl silica and most typically the packing is a porous silica having octadecylsilane (ODS) bonded to it.
Work has recently been done on improving the efficiency of ODS bonded silica packings. See, for example, Marshall et al, "Synthesis of L C Reversed Phases of Higher Efficiency by Initial Partial Deactivation of the Silica Surface," Journal of Chromatograhy Science, Vol. 22., June 1984, pp. 217-220, where it is suggested that pretreating silica with a small amount of end-capping reagent (such as trimethylchlorosilane), followed by exhaustive octadecylation, yields reverse phase packings of high efficiency (narrower band widths).
Although the efficiency of such packing materials is good, they have a limited life. While ODS packings absorb the lipophilic drug substances from the sample, they also absorb proteinaceous substances which tend to interfere with fractionation of the drug substance from other materials contained in the sample. This eventually leads to a complete fouling of the chromatographic column. Therefore, it has previously been necessary to carry out a preliminary sample preparation procedure to remove the troublesome proteins.
In the most conventional way, the proteins are precipitated, the aqueous supernatant is extracted with a water-immiscible organic solvent, the organic solvent is removed from the extract by evaporation, and the analyte residue is reconstituted in mobile phase before analysis by high-pressure liquid chromatograhy (HPLC). This method is very time-consuming and cost-inefficient.
A second method currently employed involves the adsorption of analytes onto a reverse phase packing of octadecylsilane bonded to silica in a small disposable column. Although this technique can be automated, the columns can be used for only one sample because proteins remain on the packing, and as a result the technique is also cost-inefficient for multiple samples.
In a third method, a reverse phase packing of octadecylsilane bonded to silica is introduced into a precolumn, which is separated from, but connectable to, an analytical column by a switching valve arrangement. Serum samples are injected directly into the precolumn, where the proteins are denatured and accumulated, and the deproteinated analyte solution is passed into the analytical column for fractionation. After approximately three injections, the precolumn must be backflushed to remove the protein residue. This interruptive backflush is time-inefficient for a large number of samples. Furthermore, the octadecylsilane packing eventually deteriorates because proteins cannot be completely removed therefrom.
Accordingly, for reverse phase liquid chromatography it would be desirable to have a packing material which is less protein adsorptive.
From the standpoint of size exclusion chromatography, it would be desirable to have a packing material wherein the hydrophilic bonded phase forms a dense saturated coverage of the surface, but does not form multiple polymeric layers ("multilayers"). Commercial high performance size-exclusion chromatography columns typically have packing materials comprising a porous silica support with a diol bonded phase, most typically glycerylpropylsilane. See, e.g., Pfannkoch et al, "Characterization of Some Commercial High Performance Size-Exclusion Chromatography Columns for Water-Soluble Polymers", Journal of Chromatographic Science, Vol. 18, September 1980, pp. 430-441. However, diol bonded phases tend to provide a thick, multilayered coverage when deposited from aqueous solutions.
It is known that thick multilayered bonded phases show decreased access by solutes and therefore decreased efficiency. See, e.g., Kirkland, J. "High Speed Liquid-Partition Chromatography With Chemically Bonded Organic Stationary Phases," Journal of Chromatographic Science, Vol. 9, April 1971, pp. 206-214. For that reason, typically the diol bonded phase is applied to the porous silica support under conditions in which less than saturating amounts are immobilized on the surface,typically in amounts of less than 1.0 molecules per square nanometer (i.e. .ltoreq.1.0 m/nM.sup.2). See, Regnier et al, "Glycerolpropylsilane Bonded Phases in the Steric Exclusion Chromatography of Biological Macromolecules", Journal of Chromatographic Science, Vol. 14, July 1976, pp. 316-320. However, such "light" applications result in a sparce bonded phase which can also lead to column inefficiency.
Accordingly, the need also exists for a size exclusion chromatography packing material with a dense but not multilayered diol bonded phase.